Neurobiology of Disease 146 (2020) 105139 Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi Review The epitranscriptome in stem cell biology and neural development T ⁎ Caroline Vissersa,b, Aniketa Sinhab, Guo-li Mingc,d,e,f, Hongjun Songc,d,e,g, a Biochemistry, Cellular and Molecular Biology Program, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA b Department of Biochemistry and Biophysics, Department of Psychiatry, University of California at San Francisco, San Francisco, CA 94158, USA c Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA d Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA e Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA f Department of Psychiatry, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA g The Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA ARTICLE INFO ABSTRACT Keywords: The blossoming field of epitranscriptomics has recently garnered attention across many fields by findings that Epitranscriptome chemical modifications on RNA have immense biological consequences. Methylation of nucleotides inRNA, m6A 6 6 1 including N6-methyladenosine (m A), 2-O-dimethyladenosine (m Am), N1-methyladenosine (m A), 5-methyl- Stem cells cytosine (m5C), and isomerization of uracil to pseudouridine (Ψ), have the potential to alter RNA processing Brain development events and contribute to developmental processes and different diseases. Though the abundance and roles of Brain disorders some RNA modifications remain contentious, the epitranscriptome is thought to be especially relevant instem cell biology and neurobiology. In particular, m6A occurs at the highest levels in the brain and plays major roles in embryonic stem cell differentiation, brain development, and neurodevelopmental disorders. However, studies in these areas have reported conflicting results on epitranscriptomic regulation of stem cell pluripotency and mechanisms in neural development. In this review we provide an overview of the current understanding of several RNA modifications and disentangle the various findings on epitranscriptomic regulation of stemcell biology and neural development. 1. Introduction DNA and protein (Wang and Yi 2019). In fact, over 100 “epitran- scriptomic” modifications have been identified, though their biological The central dogma of biology—that information flows from DNA to consequences are largely unknown (Cantara et al. 2011; Machnicka RNA to protein—has acquired an increasing number of caveats and fine et al. 2013). A particular methylation at the N6 position of adenosine, print (He 2019). Beyond direct exceptions to the rule, like non-coding termed m6A, has garnered the most attention for its powerful role in RNAs, the details of the process are marred by questions like how much regulating mRNA processing (Roundtree et al. 2017). RNA is made from DNA, and how is this RNA processed to make the While the existence of m6A has been known for almost 50 years correct quantities of particular proteins or isomers at the correct time? (Desrosiers et al. 1974; Lavi and Shatkin 1975; Rottman et al. 1974; Wei How might the system change in response to stimuli, and what reg- et al. 1975; Wei and Moss 1977), a combination of events reinvigorated ulatory systems drive these dynamics? The first step of the process, DNA a recent interest in the field. First, Zhong et al. showed in 2008 that to RNA, unfolds into many fields, including chromatin regulation, m6A is critical for developmental processes in Arabidopsis thaliana, epigenetics, and transcription. For example, reversible chemical mod- which suggested its importance for multicellular eukaryotes, and pu- ifications are dynamically added to DNA and histones to altergene shed for the development of m6A mapping methods (Zhong et al. 2008). expression and RNA levels (Akichika et al. 2019; Guo et al. 2011; Kohli Next, the discovery of the m6A demethylases, FTO and ALKBH5, in and Zhang 2013; Strahl and Allis 2000). The next step, making protein 2011 and 2013, respectively (Jia et al. 2011; Zheng et al. 2013), sug- from RNA, has largely been focused on the regulation of translation. gested that the m6A system may be dynamic, which implies that it can However, the recent exploration of post-transcriptional modifications be used as a regulatory system to alter mRNA fate in a context-depen- on RNA has shown that RNA is much more than a middleman between dent manner. Concurrently, antibody-based m6A mapping techniques ⁎ Corresponding author at: Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail address: [email protected] (H. Song). https://doi.org/10.1016/j.nbd.2020.105139 Received 7 April 2020; Received in revised form 9 October 2020; Accepted 11 October 2020 Available online 13 October 2020 0969-9961/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). C. Vissers, et al. Neurobiology of Disease 146 (2020) 105139 were developed and showed that m6A additions to mRNA are non- cytoplasm. However, the in vivo activity of FTO as an m6A demethylase random, thus pushing biological interest forward and providing tech- has recently been questioned, with the suggestion that it may instead 6 6 niques to understand the molecular mechanisms of m A in the cell act on m Am (Engel et al. 2018; Koh et al. 2019; Linder et al. 2015; (Dominissini et al. 2013; Meyer et al. 2012). In this review, we first Mauer et al. 2017; Schwartz et al. 2014b). On the other hand, one re- 6 6 1 introduce several epitranscriptomic marks that have garnered the most cent study reported that FTO can demethylate m A, m Am, and m A attention over recent years, with a focus on m6A. We then discuss the depending on the nuclear or cytoplasmic localization (Wei et al. 2018). major advancements in our understanding of the epitranscriptome in Yet another study argued that since loss of FTO has little effect on cy- 6 6 stem cell biology, especially embryonic stem cells (ESCs) and induced toplasmic mRNA m A or m Am levels, FTO likely functions primarily in pluripotent stem cells (iPSCs). We also touch on how the epitran- the nucleus. They further showed that methylation on small nuclear scriptome itself is regulated in embryonic stem cells. In addition, we RNAs (snRNAs) is a substrate for FTO demethylation (Mauer et al. review the role of the epitranscriptome in cortical and cerebellar de- 2019). The specificity of FTO remains a major hurdle in the fieldof velopment, as well as in adult neurogenesis. Finally, we discuss the role m6A; confirming its target is critically important so as not tomis- of the epitranscriptome in neurological disorders. attribute a phenotype or biological function to the wrong epitran- scriptomic mark. Finally, full knockouts of either ALKBH5 or FTO are 2. RNA modifications of particular interest not lethal in mice, though they appear to be particularly important in the cellular stress responses (Cao, 2019; Engel et al. 2018; Ma et al. 2.1. N6-methyladenosine: m6A 2018). The highly variable functions of m6A can be attributed to its many On a global level, 0.2 to 0.5% of all adenines are m6A modified distinct reader proteins. The central group of readers is the YTH-do- (Geula et al. 2015). The highest levels occur in the brain, where up to main-containing family of proteins, which bind directly to m6A. These 30% of all transcripts are modified (Chang et al. 2017). Epitran- readers have recently been reviewed elsewhere (Patil et al. 2018; scriptomic detection technologies have been focused on m6A, making it Roundtree et al. 2017). Briefly, YTHDC1 is found in the nucleus and one of the best-studied modifications to date.6 m A occurs in various regulates splicing, while YTHDF1, YTHDF2, YTHDF3, and YTHDC2 are types of RNA, including tRNA, rRNA, non-coding RNA (ncRNA), and cytoplasmic and are thought to have distinct functions. YTHDF1 pro- mRNA. In 2012, two groups independently reported anti-m6A antibody- motes translation, YTHDF2 promotes mRNA degradation, and YTHDF3 based m6A RIP-Seq (MeRIP-Seq) techniques (Dominissini et al. 2012; seems to promote either translation or degradation in a context-specific Meyer et al. 2012). Subsequent mapping of m6A in the transcriptome manner. Finally, the binding specificity and function of YTHDC2 re- showed that it is most commonly added at a consensus sequence of main unclear and may only be functional under special cellular con- DRACH (D = A, U or G; R = G or C; H = A, U, or C). m6A is especially ditions (Patil et al. 2018). In contrast to the notion that each YTHDF enriched in the 3’UTR and around the STOP codon (Dominissini et al. reader promotes a unique fate of m6A-modified mRNA, two recent 2012; Meyer et al. 2012). While m6A does not alter Watson-Crick- studies found that YTHDF-1, 2, and 3 are jointly promote mRNA de- Franklin base pairing, it can modify protein binding and affect the gradation in cell lines (Zaccara and Jaffrey 2020) (BioRxiv: doi: https:// mRNA secondary structure (Farre et al. 2003; Roost et al. 2015). Many doi.org/10.1101/2020.06.03.131441). These divergent conclusions on m6A-binding proteins, or readers, have been identified. Individual the function of m6A reader proteins underscore the vital need for ad- readers confer unique downstream fates on m6A-modified mRNA, in- ditional research into reader protein function and regulation. cluding altered mRNA stability, translation, localization, and splicing. 6 6 m A methylation patterns in the transcriptome appear to be cell/tissue- 2.2.